Gram-negative bacterial pathogens, including those that grow intracellularly, are known to secrete a variety of virulence molecules into the surrounding environment at specific points during infection. Such secreted proteins are important tools for Gram-negative pathogens to manipulate their host environment to allow growth in vivo and elude the host immune response. To date, there have been no secreted proteins of Francisella tularensis implicated in the pathogenesis of this important biothreat, nor have any studies directly addressed this question. Thus, this proposal will combine proteomic and genomic approaches as well as in situ and in vivo infection models to characterize the role of exoproteins expressed during F. tularensis infection. A broad and thorough analysis of proteins secreted by the highly virulent F. tularensis Schu4 into the extracellular milieu of may lead to a significant advancement in our understanding the pathogenesis of this pathogen. The Specific Aims of the project are: (1) To characterize the extracellular proteome of the virulent F. tularensis Schu4 strain grown under different conditions relevant to infection in vivo; (2) To identify the complete open reading frames (ORF's) encoding each secreted extracellular protein (Exp) characterized in Aim 1; (3) To determine the role of each F. tularensis Schu4 secreted extracellular protein (Exp) for in vitro growth in human macrophages and for virulence in murine models of tularemia; (4) To establish whether secreted extracellular proteins (Exp) of F. tularensis Schu4 represent targets for immunoprophylaxis or therapeutic treatment during early stages of tularemia. These aims will have strong relevance to the overall goals of the P01 application and close collaborations with the other five P01 investigators will markedly enhance the success of this project. These combined studies could lead to a better understand the pathogenesis of this organism, leading to the development of new diagnostic, vaccine, and intervention strategies.